DESCRIPTION (from applicant abstract) Although most amputated appendages do no regrow, mammals including humans can regrow digit tips amputated through the level on the nail. Digits amputated proximal to the nail do not regenerate. Experimental strategies to methodically investigate molecular mechanisms of nail-dependent digit-tip regrowth are limited by insufficient understanding of fundamental principals of regrowth. This study will survey four categories of tissue interactions to establish such parameters. One focus will be to determine whether regrowth represents a form of regeneration which recapitulates ontogenetic limb development or whether it is a form of hyperplasia of differentiated tissues. The distinction has implications for induced outgrowths at other levels and will be based primarily upon the patterns of proliferation and osteogenesis. Possible epithelio-mesenchymal (E-M) inductive interaction will be evaluated on the basis of the histological appearance of wound epithelium, basement membrane (BM), and mesenchyme. BM distribution will be documented using anti-laminin antibodies in regenerating (distally amputated) and non-regenerating (proximally amputated) digits. Possible extracellular matrix (ECM) suppression will be evaluated with picrosirius red staining to document collagen fiber bundle density, and with enzyme overlay membranes to document matrix protease activity, including that of matrix metalloproteinases (MMPs). The pattern and intensity will reveal whether histolysis is increased and fiber synthesis suppressed in regenerates compared with non-regenerates. Denervation experiments will determine whether digit-tip regrowth is nerve dependent. Finally, because the nail organ is know to be necessary for outgrowth of distal phalanges, it will be transplanted to proximal-level amputation sites, evaluated both in vivo and in vitro, to determine whether nail organ can induce skeletal outgrowth at other levels. Advantages of the research design include multiple analyses performed on the same piece of tissue thereby conserving animals, direct comparison of regenerating and non-regenerating appendages, and evaluation of transplantatio both in vivo and in vitro. Cumulatively, results of these studies will provide direction for more systematic studies of the molecular mechanisms of this unique form of appendage regeneration. The ultimate goal of these studies is the regulate events at amputation sites, either to suppress overgrowth or to induce appendage regrowth.